Vitamin D is converted to several biologically active metabolites in the body, and the 1,25-dihydroxyvitamin D (1,25-(OH)2D) sterol produced in the kidneys appears to be the hormonal form of the vitamin. Studies will be focused on receptor protein which facilitates retention of 1,25-(OH)2D in the intestine. The cytoplasmic receptor protein which facilitates retention of5 1,25-(OH)2D in the intestinal nucleus will be purified to homogeneity and characterized. Isolation methodology will include DNA-cellulose, ion-exchange, gel filtration and vitamin D-ligand (affinity) chromatography. Next we plan to reconstitute the purified receptor (plus hormone) with unstimulated chromatin, in vitro, and determine if increased transcription of the gene (or genes) for calcium binding protein occurs. A second objective is to utilize newly generated high specific activity 1,25-(OH)2-(3H)D3 (110 Ci/ mmol), 24,25-dihydroxy(3H)D3 (80 Ci/ mmol) and 1,24,25-trihydry(3H)D3 (80 Ci/ mmol) to search for possible new target organs for the hormone and the less well understood 24-hydroxylated D-vitamins. Standard binding techniques, along with a new DNA-cellulose assay, will be employed to detect putative receptors in bone, parathyroid, kidney, pancreas and pituitary tissue. Because all of the above steroids are labeled with tritium at carbons number 23 and number 24, their in vitro metabolism via side chain oxidation can be monitored by tritiated H2O formation. A final goal will be to develop a readioligand binding assay for circulating 1,24,25-(OH)3D and couple it with existing assays for 25-(OH)D, 24,25(OH)2D and 1,25(OH)2D to monitor the complete vitamin D metabolite profile in blood. This assay will be used in experimental animals undergoing "mineral stress" and in specimens of plasma from humans with disorders of mineral and bone metabolism. Special attention will be paid to patients (before and after 1,25-(OH)2D3 treatment) with renal osteodystrophy, vitamin D-dependent rickets, vitamin D-resistant rickets and tumor induced osteomalacia.